Osteoclasts, the sole bone-resorbing cell, not only play a pivotal role in skeletal development and maintenance but are also implicated in the pathogenesis of various bone disorders including postmenopausal osteoporosis and tumor bone metastasis (Teitelbaum, 2000; Raisz, 2005; Mundy, 2002). Osteoclasts are multinucleated giant cells that differentiate from mononuclear cells of the monocyte/macrophage lineage (Teitelbaum, 2000), thus involving both dramatic phenotypic changes and reprogramming of gene expression. Osteoclastogenesis requires two essential factors: the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL) (Suda et al., 1999; Teitelbaum, 2000; Boyle et al., 2003).
RANKL (also known as OPGL, ODF and TRANCE), a member of the tumor necrosis factor (TNF) family, was discovered independently by several groups (Anderson et al., 1997; Wong et al., 1997b; Lacey et al., 1998; Yasuda et al., 1998) in the late 1990s and thus far has been shown to regulate diverse physiological processes such as bone remodeling (Lacey et al., 1998; Yasuda et al., 1998), dendritic cell (DC) survival and activation (Wong et al., 1997a; Josien et al., 1999; Josien et al., 2000), T-cell activation (Kong et al., 1999; Bachmann et al., 1999), lymph node organogenesis (Kong et al., 1999; Dougall et al., 1999; Kim et al., 2000a), B-cell differentiation (Kong et al., 1999; Dougall et al., 1999), mammary gland development (Fata et al., 2000), and thermoregulation in females or fever response inflammation (Hanada et al., 2009). RANKL regulates various biological functions by binding to and activating its receptor RANK (Hsu et al., 1999), which belongs to the TNF receptor (TNFR) family (Anderson et al., 1997). RANKL also has a decoy receptor, osteoprotegerin (OPG) (Simonet et al., 1997; Tsuda et al., 1997), which inhibits RANKL function by competing with RANK for binding RANKL (Yasuda et al., 1998; Lacey et al., 1998).
In bone, RANKL and RANK play important roles in osteoclastogenesis: Mice lacking either protein develop osteopetrosis due to failure to form osteoclasts (Kong et al., 1999; Dougall et al., 1999; Li et al., 2000; Kim et al., 2000b). Consistently, mice deficient for OPG develop early onset of osteoporosis due to elevated osteoclastogenesis (Bucay et al., 1998; Mizuno et al., 1998) whereas transgenic mice over-expressing OPG exhibit osteopetrosis, resulting from a decrease in late stages of osteoclastogenesis (Simonet et al., 1997).
The discovery of the RANKL/RANK/OPG axis was soon followed by an intensive investigation of RANK-activated intracellular signaling pathways involved in the regulation of the diverse functions. The initial efforts primarily focused on TNF receptor associated factor (TRAF)-dependent pathways since RANK was identified as a member of the TNF receptor (TNFR) family (Anderson et al., 1997) and members of the TNFR family, which lack intrinsic enzymatic activity, transduce intracellular signals by recruiting various TRAFs via specific motifs in the cytoplasmic domain (Locksley et al., 2001; Chung et al., 2002). Numerous biochemical and functional studies have established that RANK contains three functional TRAF-binding sites (PFQEP (SEQ ID NO: 11) 369-373, PVQEET (SEQ ID NO: 12) 559-564 and PVQEQG (SEQ ID NO: 13) 604-609) that redundantly play a role in osteoclast formation and function (Liu et al., 2004) (Liu et al., 2005; Hsu et al., 1999; Darnay et al., 1998; Wong et al., 1998; Kim et al., 1999; Darnay et al., 1999; Galibert et al., 1998). Collectively, through these functional TRAF-binding motifs, RANK activates six major signaling pathways NF-κB, JNK, ERK, p38, NFATc1 and Akt, which play important roles in osteoclast formation, function and/or survival (Boyle et al., 2003; Liu et al., 2004; Feng, 2005).
On the other hand, several lines of evidence support that RANK may also activate a TRAF-independent signaling pathway(s) essential for osteoclastogenesis. It has been shown that TRAF6 acts as a key downstream signaling molecule for both RANK and IL-1R (Wu and Arron, 2003) and a single TRAF6-binding motif is able to promote osteoclastogenesis (Ye et al., 2002; Liu et al., 2004). However, administration of IL-1 to RANK−/− mice failed to induce any osteoclastogenesis in vivo (Li et al., 2000), indicating that an unidentified TRAF6-independent signaling pathway(s) is also required for osteoclastogenesis. Moreover, consistent with this in vivo finding, in vitro studies also demonstrated that IL-1 failed to stimulate osteoclastogenesis (Azuma et al., 2000; Kobayashi et al., 2000). Given that the TRAF independent signaling pathway(s) is most likely initiated by one or more motifs in the RANK cytoplasmic domain, a systematic structure/function study of the RANK cytoplasmic domain was carried out using a chimeric receptor approach (Xu et al., 2006). This study has led to an identification of a specific 4-a.a. RANK motif (IVVY (SEQ ID NO: 4) 535-538), which shares no homology with any of the known TRAF-binding sites but plays a crucial role in osteoclastogenesis by committing bone marrow macrophages (BMMs) to the osteoclast lineage (Xu et al., 2006). However, the precise molecular mechanism by which this RANK motif mediates the lineage commitment remains elusive.
RYBP (Ring1A and YY1 binding protein, also known as DEDAF and YEAF1, Genbank Accession No. BC080287) was initially identified as a protein interacting with the Polycomb group (PcG) proteins, Ring1A and M33, and the transcriptional factor YY1 in a two-hybrid screen and shown to mediate transcriptional repression in reporter assays (Garcia et al., 1999). It was later shown that RYBP also interacts with several members of the E2F family of transcription factors (Trimarchi et al., 2001; Schlisio et al., 2002), the transcriptional factor E4TF1/hGABP (Sawa et al., 2002) and ubiquitinated H2A (Arrigoni et al., 2006), a Ring1A/Ring1B-dependent chromatin mark associated with transcriptional repression (Li et al., 2007). RYBP knockout mice exhibited embryonic lethality, revealing its essential role in development (Pirity et al., 2005). In addition to embryonic lethality, either loss- or gain-of-function experiments revealed other developmental alterations including defects in neural tube closure and formation of anterior eye structures (Pirity et al., 2005; Gonzalez et al., 2008). Intriguingly, RYBP was also identified in an independent two-hybrid screen as a protein interacting with death effector domain (DED)-containing proteins such as FADD, procaspase 8, and procaspase 10 and thus named differently as the death effector domain-associated factor (DEDAF) (Zheng et al., 2001). Moreover, RYBP has other interacting partners with roles in apoptosis, the viral apoptin protein and Hippi (Danen-van Oorschot et al., 2004; Stanton et al., 2007). Consistent with a proapoptotic function for RYBP, over-expression of RYBP in cell lines promote apoptosis (Zheng et al., 2001; Danen-van Oorschot et al., 2004). A recent study has revealed that RYBP interacts with MDM2 to alter the MDM2-p53 interaction, resulting in stabilization of p53, and thus may act as a tumor suppressor (Chen et al., 2009).
PcG proteins were originally identified in Drosophila as repressors of Hox genes, a family of transcription factors that control the anteroposterior segmentation of the fruitfly body (Schuettengruber et al., 2007). Homologues of Drosophila PcG proteins have subsequently been identified in vertebrates and plants and shown to be implicated in cell differentiation, stem cell identity, tumorigenesis and genomic imprinting (Schwartz and Pirrotta, 2008; Schwartz and Pirrotta, 2007; Kohler and Villar, 2008). PcG proteins form three major PcG complexes termed Polycomb repressive complexes (PRC) 1, PRC2 and PhoRC. The core components of the PRC1 complex include mammalian homologues of Drosophila Polycomb (PC), Posterior Sex Combs (PSC), Polyhomeotic (PH), and dRING. Specifically, RYBP interacts with Ring1A and Ring1B, two mammalian homologues of dRING, and M33 (also known as CBX2), a mammalian homologue of PC (Garcia et al., 1999; Gecz et al., 1995). The PRC2 complex primarily includes mammalian homologues of the E(Z) H3K27 methyltransferase, SU(Z)12, and Extra sex combs (ESC). PhoRC contains the mammalian transcription factor YY1, homologous to Drosophila Pleiohomeotic (PHO). The PRC2 complex is responsible for catalyzing the tri-methylation of lysine 27 on histone 3 (histone H3K27me3) in the PcG target genes, which is recognized by the PRC1 complex through the mammalian homologues of Drosophila PC (Cao and Zhang, 2004). Despite the ability of RYBP to associate with PcG proteins, the functional significance of the interaction in the regulation of PcG target genes is still unknown.